Author Correction: Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys.

Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-ß-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.

Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.

Validation of the O3DY French Version (O3DY-F) for the Screening of Cognitive Impairment in Community Seniors in the Emergency Department.

BACKGROUND: It is recommended that older patients undergo systematic mental status screening when presenting to the emergency department (ED). However, the tools available are not necessarily adapted to the ED environment, therefore, quicker and easier tools are needed. OBJECTIVES: The purpose of this study is to validate the Ottawa 3DY-French (O3DY-F) Scale as a screening tool for delirium and cognitive impairment in a French-speaking cohort. METHOD: This multicenter prospective study was conducted in four hospitals across the province of Quebec. Inclusion criteria were: age ≥ 65 years, ED stay ≥ 8 h, awaiting admission to a care unit, and independent or semi-independent in their daily living activities. Cognitive status was assessed during the initial interview using the Telephone Interview for Cognitive Screening-modified (TICS-m) and the O3DY-F scale. Comparisons were made between the O3DY-F and the TICS-m and Confusion Assessment Method (CAM) to assess the sensitivity and specificity of the O3DY-F for the detection of cognitive impairment and delirium. RESULTS: A total of 313 patients were included in this study, 139 of which had a positive O3DY-F. When compared with the CAM, the O3DY-F had a sensitivity of 84.2% (95% confidence interval [CI] 60.4-96.6) and a specificity of 58.2% (95% CI 52.3-63.9) for the detection of prevalent delirium. The O3DY-F had a sensitivity of 76.2% (95% CI 66.7-84.8) and a specificity of 67.6% (95% CI 61.0-73.6) for cognitive impairment (defined as a TICS-m < 27). CONCLUSION: The O3DY-F is a useful and effective tool to screen for delirium and undetected cognitive impairment among a French-speaking cohort in the ED.

Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

Quantification of methanol in the presence of ethanol by selected ion flow tube mass spectrometry.

The quantification of trace compounds in alcoholic beverages is a useful means to both investigate the chemical basis of beverage flavor and to facilitate quality control during the production process. One compound of interest is methanol which, due to it being toxic, must not exceed regulatory limits. The analysis of headspace gases is a desirable means to do this since it does not require direct sampling of the liquid material. One established means to conduct headspace analysis is selected ion flow tube mass spectrometry (SIFT-MS). The high concentration of ethanol present in the headspace of alcoholic drinks complicates the analysis, however, via reacting with the precursor ions central to this technique. We therefore investigated whether methanol could be quantified in the presence of a large excess of ethanol using SIFT-MS. We found that methanol reacted with ionized ethanol to generate product ions that could be used to quantify methanol concentrations and used this technique to quantify methanol in beverages containing different quantities of ethanol. We conclude that SIFT-MS can be used to quantify trace compounds in alcoholic beverages by determining the relevant reaction chemistry.

Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation.

Autoimmunity and macrophage recruitment into the central nervous system (CNS) are critical determinants of neuroinflammatory diseases. However, the mechanisms that drive immunological responses targeted to the CNS remain largely unknown. Here we show that fibrinogen, a central blood coagulation protein deposited in the CNS after blood-brain barrier disruption, induces encephalitogenic adaptive immune responses and peripheral macrophage recruitment into the CNS leading to demyelination. Fibrinogen stimulates a unique transcriptional signature in CD11b(+) antigen-presenting cells inducing the recruitment and local CNS activation of myelin antigen-specific Th1 cells. Fibrinogen depletion reduces Th1 cells in the multiple sclerosis model, experimental autoimmune encephalomyelitis. Major histocompatibility complex (MHC) II-dependent antigen presentation, CXCL10- and CCL2-mediated recruitment of T cells and macrophages, respectively, are required for fibrinogen-induced encephalomyelitis. Inhibition of the fibrinogen receptor CD11b/CD18 protects from all immune and neuropathologic effects. Our results show that the final product of the coagulation cascade is a key determinant of CNS autoimmunity.

Hypoxia Inducible Factor-1α in Astrocytes and/or Myeloid Cells Is Not Required for the Development of Autoimmune Demyelinating Disease

Hypoxia-like tissue alterations, characterized by the upregulation of hypoxia-inducible factor-1α (HIF-1α), have been described in the normal appearing white matter and pre-demyelinating lesions of multiple sclerosis (MS) patients. As HIF-1α regulates the transcription of a wide set of genes involved in neuroprotection and neuroinflammation, HIF-1α expression may contribute to the pathogenesis of inflammatory demyelination. To test this hypothesis, we analyzed the effect of cell-specific genetic ablation or overexpression of HIF-1α on the onset and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. HIF-1α was mainly expressed in astrocytes and microglia/macrophages in the mouse spinal cord at the peak of EAE. However, genetic ablation of HIF-1α in astrocytes and/or myeloid cells did not ameliorate clinical symptoms. Furthermore, conditional knock-out of Von Hippel Lindau, a negative regulator of HIF-1α stabilization, failed to exacerbate the clinical course of EAE. In accordance with clinical symptoms, genetic ablation or overexpression of HIF-1α did not change the extent of spinal cord inflammation and demyelination. Overall, our data indicate that despite dramatic upregulation of HIF-1α in astrocytes and myeloid cells in EAE, HIF-1α expression in these two cell types is not required for the development of inflammatory demyelination. Despite numerous reports indicating HIF-1α expression in glia, neurons, and inflammatory cells in the CNS of MS patients, the cell-specific contribution of HIF-1α to disease pathogenesis remains unclear. Here we show that although HIF-1α is dramatically upregulated in astrocytes and myeloid cells in EAE, cell-specific depletion of HIF-1α in these two cell types surprisingly does not affect the development of neuroinflammatory disease. Together with two recently published studies showing a role for oligodendrocyte-specific HIF-1α in myelination and T-cell-specific HIF-1α in EAE, our results demonstrate a tightly regulated cellular specificity for HIF-1α contribution in nervous system pathogenesis.

Early detection of thrombin activity in neuroinflammatory disease.

Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression.

Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation.

Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis. However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown. Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation. Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo. Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia. Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage. Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease.

Serotonin and dopamine striatal innervation in Parkinson's disease and Huntington's chorea.

In contrast to our vast knowledge of the dopamine (DA) system, much less is known about the involvement of serotonin (5-HT) in neurodegenerative diseases affecting the basal ganglia. Therefore, we designed a study that aimed at characterizing the status of the striatal DA and 5-HT systems in patients who suffered from either Parkinson's (PD) or Huntington's disease (HD), compared to age-matched controls. Antibodies against tyrosine hydroxylase (TH) and 5-HT transporter (SERT) were used as markers of DA and 5-HT axonal profiles, respectively. The density and pattern of TH+ and SERT + innervation were determined by optical density measurements as well as by direct stereological estimates of labeled axon varicosities. The results reveal a significant decrease in TH immunoreactivity and TH + axon terminals throughout the striatum in both PD and HD, whereas the intensity of SERT immunostaining and the density of SERT + axon varicosities were found to be slightly increased in the striatum of PD and HD patients compared to controls. These findings reveal that the nigrostriatal DA system is significantly impaired in both PD and HD compared to the striatal 5-HT innervation, which is slightly increased in these two conditions. The striatal 5-HT augmentation observed in PD might be the result of a neural mechanism designed to compensate for DA denervation, whereas the marked atrophy of the striatum might explain the increase in the 5-HT innervation noted in HD. These findings underline the importance of the complex interplay between DA and 5-HT striatal afferents in the elaboration of appropriate motor behaviour.

Intense dopamine innervation of the subventricular zone in Huntington's disease.

Dopamine exerts a robust promoting effect on adult neurogenesis. Here, we report the presence of an intense dopamine (tyrosine hydroxylase immunoreactive) zone along the ventricular border of the caudate nucleus in patients with Huntington's disease, but not in age-matched controls. This thin (150-400 microm) paraventricular zone was composed of numerous small and densely packed dopamine axon varicosities and overlapped the deep layers of the subventricular zone. Immunoreactivity in the paraventricular zone was 50% higher than in adjacent striatal areas. This intense dopamine zone concurs with the striking increase of neurogenesis noted in the subventricular zone of Huntington's disease patients and indicates that dopamine might play a crucial role in intrinsic mechanisms designed to compensate for the massive striatal neuronal losses that occur in Huntington's disease.

Thinking the voice: neural correlates of voice perception.

The human voice is the carrier of speech, but also an "auditory face" that conveys important affective and identity information. Little is known about the neural bases of our abilities to perceive such paralinguistic information in voice. Results from recent neuroimaging studies suggest that the different types of vocal information could be processed in partially dissociated functional pathways, and support a neurocognitive model of voice perception largely similar to that proposed for face perception.

A "voice inversion effect?".

Voice is the carrier of speech but is also an "auditory face" rich in information on the speaker's identity and affective state. Three experiments explored the possibility of a "voice inversion effect," by analogy to the classical "face inversion effect," which could support the hypothesis of a voice-specific module. Experiment 1 consisted of a gender identification task on two syllables pronounced by 90 speakers (boys, girls, men, and women). Experiment 2 consisted of a speaker discrimination task on pairs of syllables (8 men and 8 women). Experiment 3 consisted of an instrument discrimination task on pairs of melodies (8 string and 8 wind instruments). In all three experiments, stimuli were presented in 4 conditions: (1) no inversion; (2) temporal inversion (e.g., backwards speech); (3) frequency inversion centered around 4000 Hz; and (4) around 2500 Hz. Results indicated a significant decrease in performance caused by sound inversion, with a much stronger effect for frequency than for temporal inversion. Interestingly, although frequency inversion markedly affected timbre for both voices and instruments, subjects' performance was still above chance. However, performance at instrument discrimination was much higher than for voices, preventing comparison of inversion effects for voices vs. non-vocal stimuli. Additional experiments will be necessary to conclude on the existence of a possible "voice inversion effect."